Cryptosporidium (Cp) is a ubiquitous waterborne pathogen that causes diarrheal disease worldwide. This pathogen causes life-threatening diarrheal disease in immunocompromised hosts, particularly AIDS patients. To date there is no effective treatment or vaccine for Cp. Studies suggest that Cp relies on mucin- like glycoproteins to attach to and invade intestinal epithelial cells. These mucin antigens are also targets of protective cellular immune responses. However, investigating the role of these antigens in host cell invasion and immune recognition is hampered by the difficulty of studying this parasite which cannot be genetically manipulated or propagated in vitro. To address the need for an expression system that mimics post-translational modifications of the native Cp mucins, we expressed Cp glycoproteins in the related apicomplexan Toxoplasma gondii (Tg). Our previous studies have demonstrated that Tg-expressed Cp mucins are glycosylated, proteolytically processed and localized similarly to the native glycoprotein. However, size discrepancies between the native and Tg- expressed antigens suggests that there may be subtle differences in glycosylation and/or proteolytic processing. In this application we propose to compare the post-translational modifications of a native and Tg- expressed Cp mucin antigen, and determine if the Tg-expressed antigen functions similarly to the native antigen in assays of antigen function and immune recognition. For these studies we will use the Cp mucin antigen, CpMuc4. We hypothesize that the Tg expression system can be employed to produce recombinant Cp glycoprotein antigens that will effectively mimic the functions and immunogenicity of the native antigen. The specific aims are: 1. To explore and compare the post-translational modifications of the native and Tg-rCpMuc4. In these studies, we will investigate proteolytic processing and glycan structure of native CpMuc4 and Tg-rCpMuc4. 2. To evaluate the ability of Tg-rCpMuc4 to mimic the function and immunogenicity of the native antigen. These studies will compare the ability of native and Tg rCpMuc4 to bind to intestinal epithelial cells, and inhibit in vitro infection. We will evaluate humoral and cell mediated immune responses in Cp-infected mice and humans using native and Tg-rCpMuc4 as antigens. An exploratory goal of this aim will be to determine if attenuated Tg expressing CpMuc4 can be employed as a vaccine vector delivery system. These studies will provide a thorough analysis of the Tg expression system as a vehicle for producing and studying Cp mucin antigens. Results from these studies will provide preliminary data for future R01 studies on identification of specific epitopes and host cell receptors involved in sporozoite attachment and invasion and the role of mucin glycotopes in host cell recognition and immune responses. PUBLIC HEALTH RELEVANCE: Cryptosporidium (Cp) are ubiquitous water-borne parasites that cause outbreaks of gastrointestinal disease worldwide. Currently there are no vaccines or treatments for this parasite. This study investigates the use of Toxoplasma gondii to study Cp antigens that are candidates for inclusion in a vaccine for cryptosporidiosis.